Murine embryonic fibroblast cell lines differentiate into three mesenchymal lineages to different extents: new models to investigate differentiation processes.

Various diseases, injuries, and congenital abnormalities may result in degeneration and loss of organs and tissues. Recently, tissue engineering has offered new treatment options for these common, severe, and costly problems in human health care. Its application is often based on the usage of differentiated stem cells. However, despite intensive research and growing knowledge, many questions remain unresolved in the process of cell differentiation. The aim of this study was to find standardized cell models for analyzing molecular mechanisms of cell differentiation. We investigated the multipotency of three standardized murine embryonic fibroblast cell cultures using histological staining, western blotting, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Our results demonstrated that NIH-3T3 and mouse embryonic fibroblast (MEF) cells were able to differentiate into adipogenic, chondrogenic, and osteogenic lineages expressing typical differentiation markers. Interestingly, Flp-In-3T3 cells did not differentiate into any of the three mesenchymal lineages, although this cell line is genetically closely related to NIH-3T3. The results were confirmed by histological staining. Flp-In-3T3, NIH-3T3, and MEF cells have usually been used for DNA transfections, recombinant protein expression, and as "feeder cells." Unlike mesenchymal stem cells (MSCs) and mesenchymal progenitor cells (MPCs), they are easy to obtain and to expand and are less prone to change their structure and morphology, even at higher passages. Our results suggest that Flp-In-3T3, MEF, and NIH-3T3 cells are highly suitable to be used as models to analyze molecular mechanisms of cell differentiation.